Ripening of mountain papaya (Vasconcellea pubescens) and ethylene dependence of some ripening events

Moya-Leon,M.A.;Moya,M.;Herrera,R. Laboratorio de Fisiologıa Vegetal,Instituto deBiología Vegetal y Biotecnología,Universidad de Talca, Casilla 747,Talca,Chile.The first noticeable event during the ripening of mountain papaya (Vasconcellea pubescens) is the rapid degreening of the skin, followed by climacteric ethylene and flesh softening. When ethylene perception is blocked by 1-methylcyclopropene (1-MCP, 0.3 mul 1(-1) for 16 h at 20degreesC), the increase in ethylene evolution is avoided, and softening and color development are partially delayed. This indicates that ethylene perception is an absolute requirement for ethylene production during ripening and that softening and color development are only partially dependent on ethylene. Our data also suggest that the changes in pH, titratable acidity, and soluble solids are not dependent on ethylene. Mountain papaya fruit displayed the typical climacteric rise in ethylene production, reaching maximum values of 1.8-3.2 mumol kg(-1) h(-1), and so confirmed the climacteric nature of the fruit. The activity of ACC oxidase paralleled changes in ethylene production. Nevertheless, when 1-MCP treated fruit recover ethylene sensitivity, a big increase in ACC oxidase activity is observed followed by a further increase, but at a reduced level, of ethylene; the reduced ACC content suggests that in these fruit the availability of ACC is limiting ethylene biosynthesis. Finally, our results indicate that treatment of mountain papaya with 1-MCP delays the onset of the climacteric phase, and therefore can be used commercially to extend effectively the short shelf life of this fruit

Purification and biochemical characterization of 1-aminocyclopropane-1-car☐ylate oxidase from banana fruit

Moya-Leon, M.A. Departamento de Ciencias Biológicas, Facultad de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile.A novel method using PEG and ammonium sulphate was used to concentrate ACC oxidase from a crude extract prepared from the pulp of ripe fruit of banana (Musa AAA group, Cavendish subgroup). The 145-fold purification of the ACC oxidase to electrophoretic homogeneity was carried out in four chromatographic steps: hydrophobic interaction, anion exchange, chromatofocusing and gel filtration. The molecular mass of the purified enzyme was estimated to be 40 000 by gel filtration and 36 000 by SDS-PAGE, indicating that the enzyme is active as a monomer. The enzyme was recognized by a polyclonal antibody directed against a recombinant polypeptide derived from the tomato ACC oxidase, showing that the banana enzyme shares immunogenic epitopes with ACC oxidases from other fruits. An isoelectric point at pH 4.9 was estimated by chromatofocusin

Molecular docking simulation analysis of alcohol acyltransferases from two related fruit species explains their different substrate selectivities

Herrera, R (reprint author), Univ Talca, Lab Fisiol Vegetal & Genet Mol, Inst Biol Vegetal & Biotecnol, Casilla 747, Talca, Chile.Tropical papaya (Carica papaya) and mountain papaya (Vasconcellea pubescens) fruits are characterised for their strong and particular aroma. The aroma of both fruits is different and dominated by esters, which are synthesised by alcohol acyltransferases (AATs). The ability to produce esters is contrasting, V. pubescens (VpAAT1) being a very active enzyme towards the production of benzyl acetate, whereas C. papaya (CpAAT1) is more active towards the production of ethyl butanoate and methyl butanoate, but not benzyl acetate. In order to understand the mechanism of action at the molecular level, the structural model of CpAAT1 protein was built by comparative modelling. Conformational interaction between the protein and several ligands was carried out by molecular docking. CpAAT1 structure showed two domains connected by a large crossover loop, with a solvent channel in the centre of the structure. CpAAT1 and VpAAT1 proteins showed similar 3D structures, including their catalytic sites, but their solvent channels showed differences in size and shape. CpAAT1 solvent channel is larger, in agreement with its higher selectivity for large acyl-CoA substrates. In addition, the most favourably predicted substrate orientation in CpAAT1 was observed for methanol and butanoyl-CoA, showing a perfect coincidence with the high production rate of methyl butanoate of C. papaya fruit

Changes in antioxidant capacity during development and ripening of goldenberry (Physalis peruviana L.) fruit and in response to 1-methylcyclopropene treatment

Moya-Leon, MA (reprint author), Univ Talca, Inst Biol Vegetal & Biotecnol, Lab Fisiol Vegetal & Genet Mol, Casilla 747, Talca, ChileGoldenberry (Physalis peruviana) is a climacteric fruit and its ripening is regulated by ethylene. Reports indicate that the fruit contains high level of antioxidant compounds. To elucidate the role of ethylene on the antioxidant capacity of this fruit during ripening and storage, the effect of 1-methylcyclopropene (1-MCP, 0.2 mu L L-1), a blocker of the ethylene receptor, and ethylene (2000 mu L L-1 Ethrel) were tested. During ripening of the fruit the ethylene production and respiration rates increased constantly until the end of the process, similar to the SSC/acidity ratio. Firmness reduction started early during development and continued throughout ripening. The measurement of total antioxidant capacity revealed a high antioxidant level in unripe fruit, and a clear increment in antioxidant capacity, ascorbic acid and polyphenol contents was observed throughout ripening with maximum values at the ripe stage. Nevertheless, after harvest the antioxidant capacity was rapidly reduced during the shelf-life period (20 degrees C) and ethylene treatment increased this reduction. Significant preservation of the antioxidant capacity, vitamin C and polyphenol content was observed in 1-MCP treated fruit. The results indicate that 1-MCP treatment could be useful to preserve the high antioxidant capacity of goldenberry fruit during storage. (C) 2012 Elsevier B.V. All rights reserved

Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene

Salvatierra, A (Salvatierra, Ariel) ; Moya-Leon, MA (Alejandra Moya-Leon, Maria); Herrera, R (Herrera, Raul). Univ Talca, Inst Biol Vegetal & Biotecnol, Talca, ChileAnthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiological roles, are produced by plants in a coordinated manner during fruit development by the action of transcription factors (TFs). These regulatory proteins have either an activating or repressing effect over structural genes from the biosynthetic pathway under their control. FaMYB1, a TF belonging to the R2R3-MYB family and isolated from commercial strawberry fruit (Fragaria x ananassa), was reported as a transcriptional repressor and its heterologous over-expression in tobacco flowers suppressed flavonoid-derived compound accumulation. FcMYB1, an ortholog of FaMYB1 isolated from the white Chilean strawberry (Fragaria chiloensis ssp. chiloensis f. chiloensis), showed higher transcript levels in white (F. chiloensis) than in red (F. x ananassa cv. Camarosa) fruits. In order to assess its contribution to the discolored phenotype in F. chiloensis, FcMYB1 was transiently down-regulated in planta using an RNAi-based approach. Quantitative real-time PCR on FcMYB1 down-regulated fruits resulted an up-regulation of anthocyanidin synthase (ANS) and a strong repression of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) transcript accumulation. In addition, these fruits showed increased concentrations of anthocyanins and undetectable levels of flavan 3-ols. Altogether, these results indicate a role for FcMYB1 in regulation of the branching-point of the anthocyanin/PA biosynthesi

Genetic Variation in a Chilean Endangered Endemic: Gomortega keule (molina) Baillon

Herrera, R.; Arias,M.; Maria A. Moya-leon, M.A.; Penailillo, P. and Caligari, Peter D. S. Instituto Biología Vegetal y Biotecnología, Universidad de Talca, 2 Norte 685, Talca, Chile.Gomortega keule (Molina) Baillon is an endangered, rare species, the only representative of its genus, and endemic to Central Chile. Populations of this tree are now fragmented and few individuals can be found in any of them. Genetic diversity was studied in 33 individuals from three populations in Cauquenes, a coastal mountain area (35°58'S-72°41'W). Fifteen InterSimple Sequence Repeat primers were used to determine the degree of similarity between and within populations. This revealed that 30% of the variation exhibited was between populations while 70% was within; nevertheless individuals were clearly clustered in a pattern which reflected a narrow base of diversity. Three other species from the Laurales order were used in order to provide an external reference as to the degree of diversity. In addition, an external wild population from the native species, Peumus boldus, was used to verify the utility of the markers. We show that the primers are effective in quickly giving an estimate of the degree of diversity of a population, thus giving important topical information relevant to preserving endangered species. Aspects of the conservation and management policy for the species in order to maintain the remaining populations and to preserve the genetic resources are discussed

Structural analysis of the alcohol acyltransferase protein family from Cucumismelo shows that enzyme activity depends on an essential solvent channel

Galaz, S (Galaz, Sebastian)[ 1 ] ; Morales-Quintana, L (Morales-Quintana, Luis)[ 1 ] ; Moya-Leon, MA (Alejandra Moya-Leon, Maria)[ 1 ] ; Herrera, R (Herrera, Raul)[ 1 ]Alcohol acyltransferases (AAT) play a key role in ester biosynthesis. In Cucumismelo var.cantalupensis, AATs are encoded by a gene family of four members (CmAAT14). CmAAT1, CmAAT3 and CmAAT4 are capable of synthesizing esters, with CmAAT1 the most active. CmAAT2 is inactive and has an Ala268 residue instead of a threonine which is present in all other active AATs, although the role of this residue is still unclear. The present work aims to understand the molecular mechanism involved in ester biosynthesis in melon fruit and to clarify the importance of the Ala268 residue. First, structural models for each protein were built by comparative modelling methodology. Afterwards, conformational interaction between the protein and several ligands, alcohols and acyl-CoAs was explored by molecular docking and molecular dynamics simulation. Structural analysis showed that CmAATs share a similar structure. Also, well-defined solvent channels were described in the CmAATs except for CmAAT2 which does not have a proper channel and instead has a small pocket around Ala268. Residues of the catalytic HxxxD motif interact with substrates within the solvent channel, with Ser363 also important. Strong binding interaction energies were described for the best substrate couple of each CmAAT (hexyl-, benzyl- and cinnamyl-acetate for CmAAT1, 3 and 4 respectively). CmAAT1 and CmAAT2 protein surfaces share similar electrostatic potentials; nevertheless the entrance channels for the substrates differ in location and electrostatic character, suggesting that Ala268 might be responsible for that. This could partly explain the major differences in activity reported for these two enzymes. Database Structural data have been deposited in the Protein Model Data Bank under the following accession numbers: CmAAT1 (PM0078514); CmAAT2 (PM0078515); CmAAT3 (PM0078516); CmAAT4 (PM0078517

Changes in the mRNA abundance of FcXTH1 and FcXTH2 promoted by hormonal treatments of Fragaria chiloensis fruit

Opazo, MC (Opazo, M. C.)[ 1 ] ; Lizana, R (Lizana, R.)[ 1 ] ; Pimentel, P (Pimentel, P.)[ 1 ] ; Herrera, R (Herrera, R.)[ 1 ] ; Moya-Leon, MA (Moya-Leon, M. A.)[ 1 ] Univ Talca, Inst Biol Vegetal & Biotecnol, Lab Fisiol Vegetal & Genet Mol, Talca, ChileChilean strawberry (Fragaria chiloensis) is a berry fruit with excellent organoleptic properties, however its fast softening limits its commercialization. Fruit softening is related to cell wall degradation and several related enzymes have been studied in strawberries, including xyloglucan endotransglycosylase/hydrolase (XTH). Two XTH genes have been identified in F. chiloensis, FcXTH1 and FcXTH2. FcXTH1 transcripts increased during fruit growth and softening, while FcXTH2 transcripts were high in vegetative tissues. XTH gene expression seems to be regulated in plants by different factors including hormones. To test their responsiveness, fruit were treated with auxin, abscisic acid, gibberellins and ethylene, and the expression of FcXTH isoforms was analysed. qPCR analysis revealed a significant variation in the expression of FcXTH genes after hormone treatments, showing an activator effect of auxins and GA(3) on both FcXTH genes, an inhibitory effect of ethylene on FcXTH2, and an inductive effect of ABA on FcXTH1 gene expression. FcXTH1 and FcXTH2 gene promoters were cloned and in silico analysed to reveal putative cis elements. Different regulatory elements responding to hormones were found in each promoter sequence. The changes in expression in response to hormone treatments are consistent with the regulatory elements described in the promoter sequences of FcXTH genes. (C) 2012 Elsevier B.V. All rights reserved

1-MCP treatment preserves aroma quality of ‘Packham's Triumph’ pears during long-term storage

Moya-Leon, M.A.; Vergara, M. Laboratorio de Fisiología Vegetal, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile. Bravo,C.; Montes, M.E. ; Moggia, C.Centro de Pomáceas, Facultad de Ciencias Agrarias, Universidad de Talca, Casilla 747, Talca, Chile.The effects of 1-MCP and CA storage in aroma production and consumer acceptance, were investigated during the long-term storage of ‘Packham‘s Triumph’ pears. Pears were harvested 144 (Harvest 1) and 157 days (Harvest 2) after full bloo